CN106754481A - The method and related culture medium of the azotobacter strain of resistance to high salt are screened from ice dish - Google Patents
The method and related culture medium of the azotobacter strain of resistance to high salt are screened from ice dish Download PDFInfo
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Abstract
Method the present invention relates to screen the azotobacter strain of resistance to high salt from ice dish, comprises the following steps：(1) bacterium inclined-plane solid medium, is prepared；(2) ice dish plant sample, is prepared；(3), bacterial strain flat board coating culture；(4), bacterial strain inclined-plane culture；(5), strain isolation；(6), bacterial strain stabilization grows.Additionally, further relating to the Growth N fixation culture medium wanted needed for the bacterium inclined-plane solid medium wanted needed for strain culturing and strain growth.The present invention can improve the efficiency of salt-enduring strain screening, can not only obtain quantity more and halophilic azotobacter strain, and increased the species of salt tolerant azotobacter strain, and the biological strain resource of good fixed nitrogen is provided for saliferous soil remediation.
Description
Technical field
The invention belongs to screen biological bacterial strain for the technical field of improving salinization soil, more particularly to a kind of resistance to high salt
The method of azotobacter strain screening and related culture medium.
Background technology
The main intensive fruit vegetables crop of a relatively high in economic benefit of the vegetables of China's facility cultivation.Fruit vegetables production will
Ask liquid manure higher to put into, with the increase of Cultivation time, soil secondary salinization, microbiota can be caused unbalance etc. and asked
Topic.It is one of important factor in order of restriction crop yield, quality and sustainable development that wherein the soil salinization is saline and alkaline.Salt marsh
Change soil to be widely distributed in the whole world, the hm of whole world saline alkali land area about 9.5 hundred million2, account for the 7.6% of the land gross area.China
Saline alkali land area accounts for the 20% of national cultivated area.In recent years, the problem of the soil salinization is increasingly severe.
Using biology techniques, the quality that such as application salt tolerant biology bacterial strain improves salinization soil is urgently to be resolved hurrily at present
Hot issue.Edaphon has stronger restorative, and the microbial diversity recovered in salinization soil is to play
With the guarantee of improved soil function.Thus obtain it is good and can adapt in, survive in salinization soil in biological bacterial strain
Then seem very necessary.But, the screening efficiency of current salt-enduring strain screening technique is low, the screening bacterial strain tolerance of salinity is low and
Limitednumber, it is difficult to meet salinization soil and administer demand.
In view of the above-mentioned technological deficiency of prior art, in the urgent need to developing a kind of life screened for improving salinization soil
The method of thing bacterial strain.
The content of the invention
It is an object of the invention to overcome shortcoming present in prior art, there is provided a kind of from ice dish
The method of the screening azotobacter strain of resistance to high salt and its related culture medium in (Mesembryanthemum crystallinum L.), its
The efficiency of salt-enduring strain screening can be improved, quantity can not only be obtained more and halophilic azotobacter strain, and increased salt tolerant
The species of azotobacter strain, the biological strain resource of good fixed nitrogen is provided for saliferous soil remediation.
To achieve these goals, the present invention provides following technical scheme：One kind screens resistance to high salt nitrogen-fixing bacteria from ice dish
The method of strain, it is characterised in that comprise the following steps：
(1) bacterium inclined-plane solid medium, is prepared
Fixed nitrogen culture medium is prepared by formula as below, and the pH value of the fixed nitrogen culture medium of preparation is adjusted to 7.0~7.2, it
Afterwards, it is, to the fixed nitrogen medium sterilization prepared 25 minutes, to obtain bacterium inclined-plane and consolidate at 0.3Mpa, temperature are 115.0 DEG C in pressure
Body culture medium, the composition of the fixed nitrogen culture medium is：Glucose 10.0g, mannitol 3.0g, potassium dihydrogen phosphate 0.5g, phosphoric acid hydrogen
Dipotassium 0.6g, calcium sulfate 0.2g, epsom salt 0.01g, mixed solution 10ml, sodium chloride 10.0g, 15.0~20.0g of agar
With distilled water 1000ml；
(2) ice dish plant sample, is prepared
A) fresh ice dish sample is taken, surface sterilization is carried out, is comprised the following steps that：Using 75% 3~5min of Ethanol Treatment,
Use sterile water wash 3~5 times afterwards, then 5min is processed with 0.5% sodium hypochlorite, reuse sterile water wash 3~5 times；
B) the ice dish sample after 5g surface sterilizations is weighed, in addition sterilizing mortar, and 10ml SPSSs is added, is ground
Wear into homogenate solution A；
C) the homogenate solution A 1ml are drawn, 9ml SPSSs are added, is vibrated 1 minute, obtain 10-1Dilution it is even
Slurry solution B；
D) the dilution homogenate solution B 1ml are drawn, 9ml SPSSs are added, 10 are obtained-2Dilution homogenate solution
C；
E) the dilution homogenate solution C 1ml are drawn, 9ml SPSSs are added, 10 are obtained-3Dilution homogenate solution
D；
F) the dilution homogenate solution D 1ml are drawn, 9ml SPSSs are added, 10 are obtained-4Dilution homogenate solution
E；
G) the dilution homogenate solution E 1ml are drawn, 9ml SPSSs are added, the dilution homogenate for obtaining 10-5 is molten
Liquid F；
(3) the dilution homogenate solution D, dilution homogenate solution E and dilution homogenate solution each 0.2ml of F, are drawn respectively, plus
Enter in bacterium inclined-plane solid medium to carry out flat board coating culture, and cultivated 4~5 days at 25~28 DEG C；
(4), the picking colony from the cultivation results of step (3), selects the culture dish of 20~200 clump counts in the bacterium
Inclined-plane culture is carried out in inclined-plane solid medium, and is cultivated 4~5 days at 25~28 DEG C；
(5), the cultivation results to step (4) are separated, and obtain the azotobacter strain of resistance to high salt.
Further, wherein, the method that the azotobacter strain of resistance to high salt is screened in the dish from ice is further included：
(6), the stabilization growth of the azotobacter strain of resistance to high salt
A) Growth N fixation culture medium is prepared：Growth N fixation culture medium, and the Growth N fixation that will be prepared are prepared by formula as below
The pH value of culture medium is adjusted to 7.0~7.2, is that growth at 0.3Mpa, temperature are 115.0 DEG C to preparing is consolidated in pressure afterwards
Nitrogen medium sterilization 25 minutes, the Growth N fixation medium component is：Yeast extract 0.1g, mannitol 15.0g, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.2g, dipotassium hydrogen phosphate 0.6g, calcium sulfate 0.2g, epsom salt 0.2g, sodium chloride, mixed solution 10ml, agar
15.0~20.0g and distilled water 1000ml, also, content according to the sodium chloride is respectively 10g, 12g, 15g, 18g and 21g
And it is configured to various different Growth N fixation culture mediums；
B) azotobacter strain of resistance to high salt for obtaining the step (5) is containing 10g, 12g, 15g, 18g and 21g chlorination successively
Cultivated in the different Growth N fixation culture mediums of sodium, 4 are cultivated under the conditions of 25~28 DEG C in every kind of Growth N fixation culture medium
~5 days, the bacterial strain that final purifying is obtained was the azotobacter strain of resistance to high salt.
Further, wherein, the component of the mixed solution in the fixed nitrogen culture medium and the Growth N fixation culture medium
All it is：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate 0.025g, ferric trichloride 0.05g, biotin 0.05g, sulfuric acid
Manganese 0.02g, copper sulphate 0.008g, are settled to after 100ml, also, constant volume with 0.22 μm of degerming ability of membrane filtration with distilled water
Obtain the mixed solution.
Additionally, the present invention also provides a kind of bacterium inclined-plane solid medium for cultivating the azotobacter strain of resistance to high salt, its feature exists
In, fixed nitrogen culture medium is prepared by formula as below, and the pH value of the fixed nitrogen culture medium of preparation is adjusted to 7.0~7.2, afterwards,
Pressure is that 0.3Mpa, temperature are, to the fixed nitrogen medium sterilization prepared 25 minutes, to obtain bacterium inclined-plane solid culture at 115.0 DEG C
Base, the composition of the fixed nitrogen culture medium is：Glucose 10.0g, mannitol 3.0g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate
0.6g, calcium sulfate 0.2g, epsom salt 0.01g, mixed solution 10ml, sodium chloride 10.0g, 15.0~20.0g of agar and steaming
Distilled water 1000ml；Wherein, the component of the mixed solution is：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate
0.025g, ferric trichloride 0.05g, biotin 0.05g, manganese sulfate 0.02g, copper sulphate 0.008g, are settled to distilled water
After 100ml, also, constant volume the mixed solution is just obtained with 0.22 μm of membrane filtration is degerming.
Again, the present invention also provides a kind of azotobacter strain of resistance to high salt stabilization growth Growth N fixation culture medium, and its feature exists
In, Growth N fixation culture medium is prepared by formula as below, and the pH value of the Growth N fixation culture medium of preparation is adjusted to 7.0~7.2,
Afterwards, it is to the Growth N fixation medium sterilization prepared 25 minutes, the growth at 0.3Mpa, temperature are 115.0 DEG C in pressure
Fixed nitrogen medium component is：Yeast extract 0.1g, mannitol 15.0g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate 0.6g, sulphur
Sour calcium 0.2g, epsom salt 0.2g, 10~21g of sodium chloride, mixed solution 10ml, 15.0~20.0g of agar and distilled water
1000ml, wherein, the component of the mixed solution is：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate 0.025g, three
Iron chloride 0.05g, biotin 0.05g, manganese sulfate 0.02g, copper sulphate 0.008g are settled to 100ml with distilled water, also, fixed
After appearance the mixed solution is just obtained with 0.22 μm of membrane filtration is degerming.
Compared with prior art, the present invention has following Advantageous Effects：
First, for China's rational sampling quantity of protected vegetable culture secondary salinization problem, filtered out and be suitable for salinization soil and apply
Bacterial strain.
2nd, the pick-up rate of salt-enduring strain can be improved by culture medium of the invention, and is remarkably improved by the present invention
The salt resistance ability of bacterial strain, specifically：By the present invention, the separable resistance to salinity of bacterial strain highest for obtaining reaches 18~21%, sieve
The bacterial strain quantity of choosing is accounted for and can cultivate the 30~35% of bacterium colony, and the species through differentiating display bacterium is maintained at 5~6 classes.
3rd, it is of the invention for the improvement of salinization soil quality provides good biological strain resource.
Specific embodiment
With reference to embodiment, the present invention is further described, and the content of embodiment is not as to protection scope of the present invention
Limitation.
Method the present invention relates to screen the azotobacter strain of resistance to high salt from ice dish, it is comprised the following steps：
First, bacterium inclined-plane solid medium is prepared.Specific method is as follows：
Fixed nitrogen culture medium is prepared by formula as below, and the pH value of the fixed nitrogen culture medium of preparation is adjusted to 7.0~7.2, it
Afterwards, it is, to the fixed nitrogen medium sterilization prepared 25 minutes, to obtain bacterium inclined-plane and consolidate at 0.3Mpa, temperature are 115.0 DEG C in pressure
Body culture medium.Wherein, the composition of the fixed nitrogen culture medium is：Glucose 10.0g, mannitol 3.0g, potassium dihydrogen phosphate 0.5g, phosphorus
Sour hydrogen dipotassium 0.6g, calcium sulfate 0.2g, epsom salt 0.01g, mixed solution 10ml, sodium chloride 10.0g, agar 15.0~
20.0g and distilled water 1000ml.
Preferably, the component of the mixed solution is：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate 0.025g,
Ferric trichloride 0.05g, biotin 0.05g, manganese sulfate 0.02g, copper sulphate 0.008g, 100ml is settled to distilled water, also,
After constant volume the mixed solution is just obtained with 0.22 μm of membrane filtration is degerming.
Secondly, ice dish plant sample is prepared.Specific method is as follows：
A) fresh ice dish sample is taken, surface sterilization is carried out.Comprise the following steps that：Using 75% 3~5min of Ethanol Treatment,
Use sterile water wash 3~5 times afterwards, then 5min is processed with 0.5% sodium hypochlorite, reuse sterile water wash 3~5 times.
Wherein, the ice dish sample can be the ice dish sample from the collection of the different regions such as Hefei ,Anhui, Funan.In order to more
Effect of the invention is proved well, it is preferable that can be gathered 10 kinds of different ice dish samples from above-mentioned different regions, is respectively adopted
The method of the present invention is tested.
B) the ice dish sample after 5g surface sterilizations is weighed, in addition sterilizing mortar, and 10ml SPSSs is added, is ground
Wear into homogenate solution A.
C) the homogenate solution A 1ml are drawn, 9ml SPSSs are added, is vibrated 1 minute, obtain 10-1Dilution it is even
Slurry solution B.
D) the dilution homogenate solution B 1ml are drawn, 9ml SPSSs are added, 10 are obtained-2Dilution homogenate solution
C。
E) the dilution homogenate solution C 1ml are drawn, 9ml SPSSs are added, 10 are obtained-3Dilution homogenate solution
D。
F) the dilution homogenate solution D 1ml are drawn, 9ml SPSSs are added, 10 are obtained-4Dilution homogenate solution
E。
G) the dilution homogenate solution E 1ml are drawn, 9ml SPSSs are added, the dilution homogenate for obtaining 10-5 is molten
Liquid F.
Again, bacterial strain flat board coating culture is carried out.Specific method is as follows：
The dilution homogenate solution D, dilution homogenate solution E and dilution homogenate solution each 0.2ml of F are drawn respectively, are added to
Flat board coating culture is carried out in bacterium inclined-plane solid medium, and is cultivated 4~5 days at 25~28 DEG C.
Then bacterial strain inclined-plane culture, is carried out.Specific method is as follows：
The picking colony from above-mentioned cultivation results, selects the culture dish of 20~200 clump counts solid on the bacterium inclined-plane
Inclined-plane culture is carried out in body culture medium, and is cultivated 4~5 days at 25~28 DEG C.
Afterwards, strain isolation is carried out.Specifically, above-mentioned cultivation results are separated, obtains the azotobacter strain of resistance to high salt.
Additionally, the quality in order to improve bacterial strain, the method that the azotobacter strain of resistance to high salt is screened in the dish from ice can be carried out
Stabilization growth including the azotobacter strain of resistance to high salt.Its specific method is as follows：
A) Growth N fixation culture medium is prepared.Specific method is as follows：
Growth N fixation culture medium is prepared by formula as below, and the pH value of the Growth N fixation culture medium of preparation is adjusted to 7.0
~7.2, it is to the Growth N fixation medium sterilization prepared 25 minutes at 0.3Mpa, temperature are 115.0 DEG C in pressure afterwards.Institute
Stating Growth N fixation medium component is：Yeast extract 0.1g, mannitol 15.0g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate
0.6g, calcium sulfate 0.2g, epsom salt 0.2g, sodium chloride, mixed solution 10ml, 15.0~20.0g of agar and distilled water
1000ml.Also, it is respectively 10g, 12g, 15g, 18g and 21g according to the content of the sodium chloride and is configured to various different
Growth N fixation culture medium.
Preferably, the component of the mixed solution is：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate 0.025g,
Ferric trichloride 0.05g, biotin 0.05g, manganese sulfate 0.02g, copper sulphate 0.008g, 100ml is settled to distilled water, also,
After constant volume the mixed solution is just obtained with 0.22 μm of membrane filtration is degerming.
B) by the azotobacter strain of resistance to high salt obtained above successively containing 10g, 12g, 15g, 18g and 21g sodium chloride not
With being cultivated in Growth N fixation culture medium, cultivated 4~5 days under the conditions of 25~28 DEG C in every kind of Growth N fixation culture medium,
The bacterial strain that final purifying is obtained is the azotobacter strain of resistance to high salt.
In order to identify the property of the azotobacter strain of resistance to high salt cultivated, it is necessary to identify the azotobacter strain of resistance to high salt.
In the present invention, the following two kinds identification is carried out：
(1), Physiology and biochemistry identification：The bacterial strain of culture is carried out into the Indexs measures such as Gram's staining successively, it includes following
Step (specific method referring to primary Jie Shi identification handbooks bacterial part Holt, J.G., Kreig, N.R., Sneath, P.H.A.,
Staley,J.T.,Williams,S.T.,1994,Bergey's manual of systematic
bacteriology.Vol.1,9th Edn(Baltimore,MD:Williams&Wilkins),1935-2045)：
In aseptic operating platform, the bacterium colony of picking growth, smear is fixed.Ammonium oxalate crystal violet dye solution is dyeed 1 minute, is washed
Bottle washing；Plus iodine solution covering painting face contaminates about 1 minute, washing sucks moisture with blotting paper；Plus 95% alcohol few drops, and gently shake
It is dynamic to be decolourized, washed after 30 seconds, suck moisture；After Huang red dyeing liquor contaminates 1 minute, wash bottle washing.Spontaneously dry, under microscope
Microscopy, records result.Result shows, screens the salt-enduring strain for obtaining and be gram-positive bacteria.
(2), the PCR amplifications of the nif gene of the bacterial strain of resistance to high salt, its comprise the following steps (specific method referring to Mirza,
B.,Welsh,A.,Rasul,G.,Rieder,J.,Paschke,M.,Hahn,D.,2009,Variation in Frankia
Populations of the Elaeagnus Host Infection Group in Nodules of Six Host
Plant Species after Inoculation with Soil.Microb Ecol 58,384-393 and Tan, J.W.,
Thong,K.L.,Arumugam,N.D.,Cheah,W.L.,Lai,Y.W.,Chua,K.H.,Rahim,R.A.,
Vikineswary,S.,2009,Development of a PCR assay for the detection of nifH and
nifD genes in indigenous photosynthetic bacteria.Int.J.Hydrogen Energy 34,
7538-7541。
It is prepared by a, fluid nutrient medium
Fluid nutrient medium is prepared by formula as below, and the pH value of the fluid nutrient medium of preparation is adjusted to 7.0~7.2, it
Afterwards, it is that growth medium at 0.3Mpa, temperature are 115.0 DEG C to preparing sterilizes 25 minutes in pressure.The fluid nutrient medium
Composition is：Yeast extract 0.1g, mannitol 15.0g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate 0.6g, calcium sulfate 0.2g, seven
Water magnesium sulfate 0.2g, 10~21g of sodium chloride, mixed solution 10ml and distilled water 1000ml.
Preferably, the component of the mixed solution is：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate 0.025g,
Ferric trichloride 0.05g, biotin 0.05g, manganese sulfate 0.02g, copper sulphate 0.008g, 100ml is settled to distilled water.
The fluid nutrient medium that will be prepared is sub-packed in the triangular flask of 250ml according to 30ml volumes, is sterilized standby.
B, strain culturing
The Facultative Halophiles that will be activated carry out Shaking culture in net platform is grasped in access fluid nutrient medium, and condition of culture is：25~
28 DEG C, 200 revs/min, culture 2~3 days, it is standby.
C, DNA of bacteria are extracted
Using SDS- proteinase-K pathways, DNA is extracted, comprised the following steps that：3500r/min centrifugation 10min, collects thalline, plus
Enter 100 μ l lysozyme suspension cells, room temperature 30min；The μ l of 10%SDS 150, Proteinase K 10 μ l, 37 DEG C of water-bath 30min are added,
300ul 5mol/L sodium acetates are added to mix, ice bath 5min；10000r/min is centrifuged 10min, takes supernatant, adds double volume pre-
Cold absolute ethyl alcohol, centrifugation is drying precipitated, is dissolved in 100 μ l TE buffer solutions, standby or -20 DEG C of preservations.
D, nif gene amplification
Above-mentioned DNA of bacteria is taken, as pcr template.Specific method referring to：((Mirza,B.,Welsh,A.,Rasul,G.,
Rieder,J.,Paschke,M.,Hahn,D.,2009,Variation in Frankia Populations of the
Elaeagnus Host Infection Group in Nodules of Six Host Plant Species after
Inoculation with Soil.Microb Ecol 58,384-393 and Tan, J.W., Thong, K.L., Arumugam,
N.D.,Cheah,W.L.,Lai,Y.W.,Chua,K.H.,Rahim,R.A.,Vikineswary,S.,2009,Development
of a PCR assay for the detection of nifH and nifD genes in indigenous
Photosynthetic bacteria.Int.J.Hydrogen Energy 34,7538-7541) etc. report method.PCR is produced
Thing carries out agarose electrophoresis, detects PCR primer.Result shows that screening obtains bacterial strain has nifHDK, illustrates the bacterium of screening
Strain has nitrogen fixing capacity.
The present invention is directed to China's rational sampling quantity of protected vegetable culture secondary salinization problem, and screening is suitable for salinization soil administration
Bacterial strain.By using the method for the present invention, the separable resistance to salinity of bacterial strain highest for obtaining reaches 18~21%, the bacterium of screening
Strain number amount is accounted for can cultivate the 30~35% of fixed nitrogen bacterium colony, and the species through differentiating display bacterium is maintained at 5~6 classes.Side of the invention
Method improves the efficiency screened for salt-enduring strain at present, can not only obtain quantity more and halophilic azotobacter strain, and
The species of salt tolerant azotobacter strain is increased, the biological bacterial strain money of good fixed nitrogen is provided for saliferous soil remediation
Source.
The above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not to the present invention
Implementation method restriction.For those of ordinary skill in the field, can also make on the basis of the above description
The change or variation of other multi-forms.Here all of implementation method cannot be exhaustive.It is every to belong to skill of the invention
Obvious change that art scheme is extended out changes row still in protection scope of the present invention.
Claims (5)
1. a kind of method that the azotobacter strain of resistance to high salt is screened in dish from ice, it is characterised in that comprise the following steps：
(1) bacterium inclined-plane solid medium, is prepared
Fixed nitrogen culture medium is prepared by formula as below, and the pH value of the fixed nitrogen culture medium of preparation is adjusted to 7.0~7.2, afterwards,
Pressure is that 0.3Mpa, temperature are, to the fixed nitrogen medium sterilization prepared 25 minutes, to obtain bacterium inclined-plane solid culture at 115.0 DEG C
Base, the composition of the fixed nitrogen culture medium is：Glucose 10.0g, mannitol 3.0g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate
0.6g, calcium sulfate 0.2g, epsom salt 0.01g, mixed solution 10ml, sodium chloride 10.0g, 15.0~20.0g of agar and steaming
Distilled water 1000ml；
(2) ice dish plant sample, is prepared
A) fresh ice dish sample is taken, surface sterilization is carried out, is comprised the following steps that：Using 75% 3~5min of Ethanol Treatment, afterwards
With sterile water wash 3~5 times, then 5min is processed with 0.5% sodium hypochlorite, reuse sterile water wash 3~5 times；
B) the ice dish sample after 5g surface sterilizations is weighed, in addition sterilizing mortar, and 10ml SPSSs is added, is ground to form
Homogenate solution A；
C) the homogenate solution A 1ml are drawn, 9ml SPSSs are added, is vibrated 1 minute, obtain 10-1Dilution homogenate it is molten
Liquid B；
D) the dilution homogenate solution B 1ml are drawn, 9ml SPSSs are added, 10 are obtained-2Dilution homogenate solution C；
E) the dilution homogenate solution C 1ml are drawn, 9ml SPSSs are added, 10 are obtained-3Dilution homogenate solution D；
F) the dilution homogenate solution D 1ml are drawn, 9ml SPSSs are added, 10 are obtained-4Dilution homogenate solution E；
G) the dilution homogenate solution E 1ml are drawn, 9ml SPSSs are added, the dilution homogenate solution F of 10-5 is obtained；
(3) the dilution homogenate solution D, dilution homogenate solution E and dilution homogenate solution each 0.2ml of F, are drawn respectively, are added to
Flat board coating culture is carried out in bacterium inclined-plane solid medium, and is cultivated 4~5 days at 25~28 DEG C；
(4), the picking colony from the cultivation results of step (3), selects the culture dish of 20~200 clump counts on the bacterium inclined-plane
Inclined-plane culture is carried out in solid medium, and is cultivated 4~5 days at 25~28 DEG C；
(5), the cultivation results to step (4) are separated, and obtain the azotobacter strain of resistance to high salt.
2. the method that the azotobacter strain of resistance to high salt is screened in the dish from ice according to claim 1, it is characterised in that further wrap
Include：
(6), the stabilization growth of the azotobacter strain of resistance to high salt
A) Growth N fixation culture medium is prepared：Growth N fixation culture medium, and the Growth N fixation culture that will be prepared are prepared by formula as below
The pH value of base is adjusted to 7.0~7.2, is the Growth N fixation training at 0.3Mpa, temperature are 115.0 DEG C to preparing in pressure afterwards
Support base to sterilize 25 minutes, the Growth N fixation medium component is：Yeast extract 0.1g, mannitol 15.0g, potassium dihydrogen phosphate
0.2g, dipotassium hydrogen phosphate 0.6g, calcium sulfate 0.2g, epsom salt 0.2g, sodium chloride, mixed solution 10ml, agar 15.0~
20.0g and distilled water 1000ml, also, content according to the sodium chloride is respectively 10g, 12g, 15g, 18g and 21g and prepares
Into various different Growth N fixation culture mediums；
B) azotobacter strain of resistance to high salt for obtaining the step (5) is containing 10g, 12g, 15g, 18g and 21g sodium chloride successively
Cultivated in different Growth N fixation culture mediums, cultivated 4~5 under the conditions of 25~28 DEG C in every kind of Growth N fixation culture medium
My god, the bacterial strain that final purifying is obtained is the azotobacter strain of resistance to high salt.
3. the method that the azotobacter strain of resistance to high salt is screened from ice dish described in a claim 2, it is characterised in that the fixed nitrogen training
The component of mixed solution supported in base and the Growth N fixation culture medium is all：Sodium molybdate 0.025g, ferrous sulfate 0.001g, molybdenum
Sour sodium 0.025g, ferric trichloride 0.05g, biotin 0.05g, manganese sulfate 0.02g, copper sulphate 0.008g, are settled to distilled water
After 100ml, also, constant volume the mixed solution is just obtained with 0.22 μm of membrane filtration is degerming.
4. a kind of bacterium inclined-plane solid medium for cultivating the azotobacter strain of resistance to high salt, it is characterised in that prepare solid by formula as below
Nitrogen culture medium, and the pH value of the fixed nitrogen culture medium of preparation is adjusted into 7.0~7.2, is that 0.3Mpa, temperature are in pressure afterwards
To the fixed nitrogen medium sterilization 25 minutes prepared at 115.0 DEG C, bacterium inclined-plane solid medium is obtained, the fixed nitrogen culture medium
Composition is：Glucose 10.0g, mannitol 3.0g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 0.6g, calcium sulfate 0.2g, seven water sulphur
Sour magnesium 0.01g, mixed solution 10ml, sodium chloride 10.0g, 15.0~20.0g of agar and distilled water 1000ml；Wherein, it is described mixed
Close solution component be：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate 0.025g, ferric trichloride 0.05g, biotin
0.05g, manganese sulfate 0.02g, copper sulphate 0.008g, are settled to after 100ml, also, constant volume with 0.22 μm of filter membrane with distilled water
Filtration sterilization just the mixed solution.
5. a kind of azotobacter strain of resistance to high salt stablizes growth Growth N fixation culture medium, it is characterised in that is prepared by formula as below and given birth to
Fixed nitrogen culture medium long, and the pH value of the Growth N fixation culture medium of preparation is adjusted to 7.0~7.2, afterwards, it is in pressure
0.3Mpa, temperature are to the Growth N fixation medium sterilization prepared 25 minutes, the Growth N fixation medium component at 115.0 DEG C
For：Yeast extract 0.1g, mannitol 15.0g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate 0.6g, calcium sulfate 0.2g, seven water sulphur
Sour magnesium 0.2g, sodium chloride 10-21g, mixed solution 10ml, 15.0~20.0g of agar and distilled water 1000ml, wherein, it is described mixed
Close solution component be：Sodium molybdate 0.025g, ferrous sulfate 0.001g, sodium molybdate 0.025g, ferric trichloride 0.05g, biotin
0.05g, manganese sulfate 0.02g, copper sulphate 0.008g, are settled to after 100ml, also, constant volume with 0.22 μm of filter membrane with distilled water
Filtration sterilization just the mixed solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107896545A (en) * | 2017-11-22 | 2018-04-13 | 南充有机蔬菜工程技术中心 | A kind of method for aiding in solving the salinization of soil using the crop rotation of ice dish |
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| CN114410471A (en) * | 2021-12-24 | 2022-04-29 | 浙江大学 | Method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil and culture medium used in method |
| CN114410471B (en) * | 2021-12-24 | 2023-11-24 | 浙江大学 | Screening method of rice rhizosphere soil non-symbiotic nitrogen-fixing bacterial strain and culture medium used by same |
| CN115927506A (en) * | 2022-12-31 | 2023-04-07 | 中国科学院新疆理化技术研究所 | Preparation method of azotobacter polysaccharide for saline-alkali soil |
| CN117143784A (en) * | 2023-10-31 | 2023-12-01 | 山东普泽汇生物工程有限公司 | Method for efficiently separating endophytes |
| CN117143784B (en) * | 2023-10-31 | 2024-01-26 | 山东普泽汇生物工程有限公司 | Method for separating endophytes |
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